A method for spectral analysis of Förster resonance energy transfer (FRET) signals is presented, taking into consideration both the contributions of unpaired donor and acceptor fluorophores and the influence of incomplete labeling of the interacting partners. It is shown that spectral analysis of intermolecular FRET cannot yield accurate values of the Förster energy transfer efficiency E, unless one of the interactors is in large excess and perfectly labeled. Instead, analysis of donor quenching yields a product of the form Ef(d)p(a), where f(d) is the fraction of donor-type molecules participating in donor-acceptor complexes and p(a) is the labeling probability of the acceptor. Similarly, analysis of sensitized emission yields a product involving Ef(a). The analysis of intramolecular FRET (e.g., of tandem constructs) yields the product Ep(a). We use our method to determine these values for a tandem construct of cyan fluorescent protein and yellow fluorescent protein and compare them with those obtained by standard acceptor photobleaching and fluorescence lifetime measurements. We call the method lux-FRET, since it relies on linear unmixing of spectral components.

The common neurotransmitter serotonin controls different aspects of early neuronal differentiation, although the underlying mechanisms are poorly understood. Here we report that activation of the serotonin 5-HT 7 receptor promotes synaptogenesis and enhances synaptic activity in hippocampal neurons at early postnatal stages. An analysis of G␣ 12 -deficient mice reveals a critical role of G 12 -protein for 5-HT 7 receptor-mediated effects in neurons. In organotypic preparations from the hippocampus of juvenile mice, stimulation of 5-HT 7 R/G 12 signaling potentiates formation of dendritic spines, increases neuronal excitability, and modulates synaptic plasticity. In contrast, in older neuronal preparations, morphogenetic and synaptogenic effects of 5-HT 7 /G 12 signaling are abolished. Moreover, inhibition of 5-HT 7 receptor had no effect on synaptic plasticity in hippocampus of adult animals. Expression analysis reveals that the production of 5-HT 7 and G␣ 12 -proteins in the hippocampus undergoes strong regulation with a pronounced transient increase during early postnatal stages. Thus, regulated expression of 5-HT 7 receptor and G␣ 12 -protein may represent a molecular mechanism by which serotonin specifically modulates formation of initial neuronal networks during early postnatal development.

In the present study we analyzed the oligomerization state of the serotonin 5-HT1A receptor and studied oligomerization dynamics in living cells. We also investigated the role of receptor palmitoylation in this process. Biochemical analysis performed in neuroblastoma N1E-115 cells demonstrated that both palmitoylated and non-palmitoylated 5-HT1A receptors form homo-oligomers and that the prevalent receptor species at the plasma membrane are dimers. A combination of an acceptor-photobleaching FRET approach with fluorescence lifetime measurements verified the interaction of CFP- and YFP-labeled wild-type as well as acylation-deficient 5-HT1A receptors at the plasma membrane of living cells. Using a novel FRET technique based on the spectral analysis we also confirmed the specific nature of receptor oligomerization. The analysis of oligomerization dynamics revealed that apparent FRET efficiency measured for wild-type oligomers significantly decreased in response to agonist stimulation, and our combined results suggest that this decrease was mediated by accumulation of FRET-negative complexes rather than by dissociation of oligomers to monomers. In contrast, the agonist-mediated decrease of FRET signal was completely abolished in oligomers composed by non-palmitoylated receptor mutants, demonstrating the importance of palmitoylation in modulation of the structure of oligomers.